Project Summary Gross chromosomal rearrangements (GCRs) are mutations known to underlie many genetic diseases. The accumulation of GCRs is a type of increased genome instability seen in many cancers and likely represents a type of mutator phenotype. Some inherited cancer susceptibility syndromes result from genetic defects that cause increased accumulation of GCRs in model systems, although whether defects in these genes drive the development of sporadic cancers is less clear. While many DNA repair and damage response pathways thought to play a role in preventing genome instability have been studied, a comprehensive understanding of the genes, pathways and mechanisms that prevent genome instability is not available. Understanding these mechanisms will impact on human health for several reasons: 1) Identifying genes that suppress GCRs will provide tools to identify causes of genome instability in cancer; and 2) Genome instability is a potential therapeutic target for cancer and understanding pathways that suppress genome instability will provide a rational basis for developing novel cancer diagnostics and therapeutic approaches. The goal of this proposal is to use Saccharomyces cerevisiae to identify genes, pathways and mechanisms that suppress GCRs. Key related objectives are to identify chromosomal features and aberrant DNA repair mechanisms that contribute to the formation of GCRs and to identify human genes in which defects cause genome instability in cancer. The proposed studies will build on the results of work supported by this project that have resulted in innovative assays for studying GCRs and have identified numerous genome instability suppressing (GIS) genes and cooperating GIS genes that suppress the accumulation of GCRs in S. cerevisiae. The following lines of research will be carried out: 1) Robotic-based genetic analysis will be performed to extend the already identified GCR suppressing genetic network by incorporating data generated with hypomorphic mutations in essential genes and data on activation of DNA damage response genes in single and double mutants with and without increased genome instability; 2) Genetic studies and whole genome sequencing will be used to identify the genes and mechanisms that specifically suppress or promote GCRs mediated by the formation of DNA hairpins and by genomic features including segmental duplications and short repeated sequences; 3) The mechanistic features of selected pathways that suppress GCRs will be investigated, initially focusing on Casein Kinase II, Exonuclease 1 and RNase H2, and subsequently on DNA replication and chromosome cohesion/condensing pathways; and 4) Human homologues of the S. cerevisiae GIS genes will be used to mine available cancer genomics data sets to identify essential and non-essential genes in which defects cause genome instability in cancer. These studies will provide a comprehensive picture of the pathways and mechanisms that prevent GCRs and provide insights into the origin of genome instability in cancer.